Compounds with growth hormone releasing properties

ABSTRACT

Compounds of the formula A--B--C--D(--E) p  are used to stimulate the release of growth hormone from the pituitary.

FIELD OF INVENTION

The present invention relates to novel peptide derivatives, compositionscontaining them, and their use for treating medical disorders resultingfrom a deficiency in growth hormone.

BACKGROUND OF THE INVENTION

Growth hormone is a hormone which stimulates growth of all tissuescapable of growing. In addition, growth hormone is known to have anumber of effects on metabolic processes, e.g., stimulation of proteinsynthesis and free fatty acid mobilization and to cause a switch inenergy metabolism from carbohydrate to fatty acid metabolism. Deficiencyin growth hormone can result in a number of severe medical disorders,e.g., dwarfism.

Growth hormone is released from the pituitary. The release is undertight control of a number of hormones and neurotransmitters eitherdirectly or indirectly. Growth hormone release can be stimulated bygrowth hormone releasing hormone (GHRH) and inhibited by somatostatin.In both cases the hormones are released from the hypothalamus but theiraction is mediated primarily via specific receptors located in thepituitary. Other compounds which stimulate the release of growth hormonefrom the pituitary have also been described. For example arginine,L-3,4-dihydroxyphenylalanine (L-Dopa), glucagon, vasopressin, PACAP(pituitary adenylyl cyclase activating peptide), muscarinic receptoragonists and a synthethic hexapeptide, GHRP (growth hormone releasingpeptide) release endogenous growth hormone either by a direct effect onthe pituitary or by affecting the release of GHRH and/or somatostatinfrom the hypothalamus.

In disorders or conditions where increased levels of growth hormone isdesired, the protein nature of growth hormone makes anything butparenteral administration non-viable. Furthermore, other directly actingnatural secretagogues, e.g., GHRH and PACAP, are longer polypeptides forwhich reason oral administration of them is not viable.

The use of shorter peptides for increasing the levels of growth hormonein mammals has previously been proposed, e.g. in EP 18 072, EP 83 864,WO 89/07110, WO 89/01711, WO 89/10933, WO 88/9780, WO 83/02272, WO91/18016, WO 92/01711 and WO 93/04081.

The composition of growth hormone releasing peptides or peptidederivatives is important for their growth hormone releasing potency aswell as their bioavailability. It is therefore the object of the presentinvention to provide peptides with growth hormone releasing propertieswhich have improved properties relative to known peptides of this type.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a compound of generalformula I

    A--B --C --D(--E).sub.p                                    I

wherein p is 0 or 1;

A is hydrogen or R¹ --(CH₂)_(q) --(X)_(r) --(CH_(s))_(s) --CO--, wherein

q is 0 or an integer between 1 and 5;

r is 0 or 1;

s is 0 or an integer between 1 and 5;

R¹ is hydrogen, imidazolyl, guanidino, piperazino, morpholino,piperidino or N(R²)--

R³, wherein each of R² and R³ is independently hydrogen or lower alkyloptionally substituted by one or more hydroxyl, pyridinyl or furanylgroups; and

X, when r is 1, is --NH--, --CH₂ --, --CH═CH--, ##STR1## wherein each ofR¹⁶ and R¹⁷ is independently hydrogen or lower alkyl;

B is (G)_(t) --(H)_(u) wherein

t is 0 or 1;

u is 0 or 1;

G and H are amino acid residues selected from the group consisting ofnatural L-amino acids or their corresponding D-isomers, or non-naturalamino acids such as 1,4-diaminobutyric acid, aminoisobutyric acid,1,3-diaminopropionic acid, 4-aminophenylalanine, 3-pyridylalanine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,1,2,3,4-tetrahydronorharman-3-carboxylic acid, N-methylanthranilic acid,anthranilic acid, N-benzylglycine, 3-aminomethylbenzoic acid,3-amino-3-methyl butanoic acid, sarcosine, nipecotic acid oriso-nipecotic acid; and wherein, when both t and u are 1, the amide bondbetween G and H is optionally substituted by ##STR2## wherein Y is >C═Oor ##STR3## and R¹⁸ is hydrogen, lower alkyl or lower aralkyl; C is aD-amino acid of formula --NH--CH((CH₂)_(w) --R⁴)--CO--wherein w is 0, 1or 2; and

R⁴ is selected from the group consisting of ##STR4## each of which isoptionally substituted with halogen, lower alkyl, lower alkyloxy, loweralkylamino, amino or hydroxy;

D, when p is 1, is a D-amino acid of formula --NH--CH((CH₂)_(k)--R⁵)--CO--or, when p is 0,

D is --NH--CH((CH₂)₁ --R⁵)--CH₂ --R⁶ or --NH --CH((CH₂)_(m--R)⁵)--CO--R⁶, wherein

k is 0, 1 or 2;

I is 0, 1 or 2;

m is 0, 1 or 2;

R ⁵ is selected from the group consisting of ##STR5## each of which isoptionally substituted with halogen, alkyl, alkyloxy amino or hydroxy;and

R⁶ is piperazino, morpholino, piperidino, --OH or--N(R⁷)--R⁸, whereineach of R⁷ and R⁸ is independently hydrogen or lower alkyl;

E, when p is 1, is --NH--CH(R¹⁰)--(CH₂)_(v) --R⁹, wherein

v is 0 or an integer between 1 and 8;

R⁹ is hydrogen, imidazolyl, guanidino, piperazino, morpholino,piperidino, 1¹⁹ ##STR6## wherein n is 0, 1 or 2, and R¹⁹ is hydrogen orlower alkyl, ##STR7## wherein o is an integer from 1 to 3, orN(R¹¹)--R¹², wherein each of R¹¹ and R¹² ² is independently hydrogen orlower alkyl, or ##STR8## each of which is optionally substituted withhalogen, alkyl, alkyloxy, amino, alkylamino, hydroxy, or the Amadorirearrangement product from an amino group and a hexapyranose or ahexapyranosyl-hexapyranose and

R¹⁰, when p is 1, is selected from the group consisting of --H, --COOH,--CH₂ --R¹³, --CO--R¹³ or --CH₂ --OH, wherein

R¹³ is piperazino, morpholino, piperidino, --OH or --N(R¹⁴)--R¹⁵,wherein each of R¹⁴ and R¹⁵ is independently hydrogen or lower alkyl;

the amide bond between B and C or, when t and u are both 0, between Aand C being optionally substituted by ##STR9## wherein Y is >C═or##STR10## and R¹⁸ is hydrogen, lower alkyl or lower aralkyl, or, when pis 1, the amide bond between D and E being optionally substituted by##STR11## wherein Y and R¹⁸ are as indicated above; or apharmaceutically acceptable salt thereof.

It is believed that peptide derivatives of formula I exhibit an improvedresistance to proteolytic degradation by enzymes due to the presence ofadjacent D-amino acids in the peptide sequence, optionally combined withthe substitution of an amide bond (--CO--NH--) by ##STR12## as indicatedabove, e.g. aminomethylene (--CH₂ --NH--) and/or modification at the N-or C-terminal end of the peptide. The increased resistance toproteolytic degradation combined with the reduced size of the peptidederivatives of the invention is expected to improve theirbioavailability compared to that of the peptides suggested in the priorliterature.

In the present context, the term "lower alkyl" is intended to indicatealkyl with 1-6 carbon atoms, in particular methyl, ethyl, propyl,iso-propyl, butyl, pentyl or hexyl. The term "halogen" is intended toinclude Cl, F, Br and I. In the terms "lower alkyloxy", "lower aralkyl"an d "lower alkylamino", t he lower alkyl moiety has the meaningindicated above.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment of the compound of formula I, p is 1. Inanother preferred embodiment of the compound of formula I, A is hydrogenor, alternatively, R¹ --(CH₂), a (X)r --(CH₂), --CO--, wherein R¹ is3-imidazolyl, q is 2, r is 0 and s is 0; or wherein R¹ is NH₂, q is 1, ris 1, X is disubstituted benzene preferably substituted in the 1 and 3positions, and s is 0; or wherein R¹ is NH , q is 1, r is 1, X isdisubstituted thiophene preferably substituted in the 3 and 2 positions,and S is 0. When t is 1, G in the compound of formula I is preferablyAla, Gly, Aib, sarcosine, nipecotic acid or isonipecotic acid. When u is1, H is preferably His, Phe, Tic, Phe(4--NH ,), 3-Pyal, Gly, Ala, Sar,Pro, Tyr, Arg, Orn, 3-aminomethylbenzoic acid or D-Phe. C in thecompound of formula I is preferably D-2-naphthylalanine (D-2Nal),D-1-naphthylalanine (D-lNal), D-Phe or D-Trp. D in the compound offormula I is preferably D-Phe, D- lN a l, D-2Nal, D-Trp, 3-Pyal,D-Phe(4F), D-Tyr or Phe-NH₂.

E in the compound of formula I is preferably Lys--NH₂,NH--(2-(1-piperazino)ethyl), NH--(2-(1-morpholino)propyl),NH--(2-aminoethyl), NH--(4-aminomethylbenzyl), NH--(benzyl), Lys--OH,NH--(1-hydroxy-6-amino-2S-hexyl),NH--(2-(1-methyl-2-pyrrolidinyl)ehtyl), or ##STR13## R⁴ in the compoundof formula I is preferably 2-naphthyl. R¹ is preferably phenyl. v ispreferably 2-6, and R⁹ is NH₂, morpholinoethyl, morpholinopropyl or(1-methylpyrrolidinyl)ethyl. R' is preferably --COOH, --CH₂ --OH, --H,--CONH₂ or --CON(CH₃)₂.

Examples of specific compounds of the present invention are

H-Ala- Hisψ(CH₂ NH)D-2Nal-D-Phe-Lys-NH₂

H-Ala-Ala-D-2Nal-D-Phe-Lys-NH₂

H-His-D-2Nal-D-Phe-Lys-NH₂

(3-(4-Imidazolyl)propionyl)-D-2Nal-D-Phe-Lys-NH₂

H-D-Lys-D-2Nal-D-Phe-Lys-NH₂

H-5Apent-His-D-2Nal-D-Phe-Lys-NH₂

H-D-Ala-D-2Nal-D-Phe-Lys-NH₂

H-5Apent-D-2Nal-D-Phe-Lys-NH₂

(n-Propyl)-His-D-2Nal-D-Phe-Lys-NH₂

H-Ala-3Pyal-D-2Nal-D-Phe-Lys-NH₂

H-Ala-Phe(4-NH₂)-D-2Nal-D-Phe-Lys-NH₂

H-D-Ala-His-D-2Nal-D-Phe-Lys-NH₂

(2-(4-Imidazolyl)acetyl)-D-2Nal-D-Phe-Lys-NH₂

(3-(4-Imidazolyl)acryloyl)-D-2Nal-D-Phe-Lys-NH₂

(3-Aminomethyl benzoyl)-D-2Nal-D-Phe-Lys-NH t

(3-Aminophenylacetyl)-D-2Nal-D-Phe-Lys-NH₂

(4-Aminophenylacetyl)-D-2Nal-D-Phe-Lys-NH₂

(3-Aminocrotonoyl)-D-2Nal-D-Phe-Lys-NH₂

(4-Piperidino-carboxyl)-D-2Nal-D-Phe-Lys-NH₂

H-Ala-His-D-2Nal-D-Phe-NH₂

(H-Ala-His-D-2Nal-D-Phe-NH)hexane

6-(H-Ala-His-D-2Nal-D-Phe-NH)hexylamine

5-(H-Ala-His-D-2Nal-D-Phe-NH)pentylanaine

H-Ala-His-D-2Nal-D-Pheψ(CH ,NH)Lys-NH₂

H-Ala-His-D-2Nal-D-Phe-Lys-OH

(2S)-(H-Ala-His-D-2Nal-D-Phe-NH)-6-aminohexanol

(2-(H-Ala-His-D-2Nal-D-Phe-NH)ethyl)benzene

2-(H-Ala-His-D-2Nal-D-Phe-NH)ethylamine

4-((H-Ala-His-D-2Nal-D-Phe-NH)methyl)benzylamine

H-Ala-His-D-2Nal-D-Phe-Lys(maltosyl)-NH₂

H-Ala-His-D-2Nal-D-Phe-Phe-NH₂

H-Ala-His-D-2Nal-D-Phe-D-Phe-NH₂

H-Ala-His-D-Phe-D-Phe-Lys-NH₂

H-Ala-His-D-Trp-D-Phe-Lys-NH₂

H-His-D-2Nal-D-Trp-Lys-NH₂

H-Ala-His-D-lNal-D-Phe-Lys-NH₂

H-Ala-Phe-D-2Nal-D-Phe-Lys-NH₂

H-Ala-His-D-2Nal-D-Phe-Lys(maltosyl)-NH₂

(2R)-(H-Ala-His-D-2Nal-D-Phe-Lys-NH)-3phenylpropylamine

H-Ala-N-Me-(2-aminobenzoyl)-D-2Nal-D-Phe-Lys-NH₂

(3-(Methylaminomethyl)benzoyl)-D-2Nal-D-Phe-Lys-NH₂

(4-(Aminomethyl)benzoyl)-D-2Nal-D-Phe-Lys-NH₂

H-His-Ala-D-2Nal-D-Phe-Lys-NH₂

4-(H-Ala-His-D-2Nal-D-Phe-NH)butylamine

3-(H-Ala-His-D-2Nal-D-Phe-NH)propylamine

(3-(Dimethylaminomethyl)benzoyl)-D-2Nal-D-Phe-Lys-NH₂

(3-Amino-3-methylbutanoyl)-D-2Nal-D-Phe-Lys-NH₂

(3-Aminomethylbenzoyl)-D-hPhe-D-Phe-Lys-NH₂

(3-Aminomethylbenzoyl)ψ(CH₂ NH)D-2Nal-D-Phe-Lys-NH₂

(3-Aminomethylbenzoyl)-D-2Nal-D-hPhe-Lys-NH₂

(3-Amino-3-methylbutanoyl)-His-D-2Nal-D-Phe-Lys-NH₂

(3-Aminomethylbenzoyl)-D-2Nal-N-Bzl-Gly-Lys-NH₂

(2S)-(3-aminomethylbenzoyl) *(CH₂ NH)-D-2Nal-D-Phe-NH)-6-aminohexanol

(2S)-((3-aminomethylbenzoyl)-D-2Nal-D-Phe-NH)-6-aminohexanol

(3-Aminomethylbenzoyl)-D-2Nal-D-Thial-Lys-NH₂

(2S)-(H-Aib-His *(CH₂ NH)-D-2Nal-D-Phe-NH)-6-aminohexanol

(³ -Aminomethylbenzoyl)-D-2Nal-D-3Pyal-Lys-NH₂

(3-Aminomethylbenzoyl)-D-2Nal-D-Phe(4-F)-Lys-NH₂

(3-Aminomethylbenzoyl)-D-2Nal-D-Phe(4-OMe)-Lys-NH₂

(2-Aminomethylphenylacetyl)-D-2Nal-D-Phe-Lys-NH₂

(2-Aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂

2-(H-Aib-His-D-2Nal-D-Phe-NH)-(4-pyridyl)ethane

H-Aib-Phe-D-2Nal-D-Phe-Lys-NH₂

2-(H-Aib-His-D-2Nal-D-Phe-NH)-( 1-methyl-2-pyrrolidinyl)ethane

2-(H-Aib-His-D-2Nal-D-Phe-NH)-(4-pyridyl)ethane

H-Aib- Msψ(CH ,NH)-D-2Nal-D-Phe-Lys-OH

(3-Am inomethylbenzoyl)-D-2Nal-N-Me-D-Phe-Lys-NH₂

H-Aib-His-D-2Nal-D-Phe-Gly-NH₂

H-Aib-His-D-2Nal-D-Phe-Ala-NH₂

H-Aib-His-D-2Nal-D-Phe-Orn-NH₂

(5-Aminomethylthienyl-2-carbonyl)-D-2Nal-D-Phe-Lys-NH₂

H-Aib-His-D-2Nal-D-Phe-D-Lys-NH₂

H-Aib-His-D-2Nal-D-Phe-Dab-NH₂

H-Aib-His-D-2Nal-D-Pheψ(CH₂ NH)-Lys-NH₂

H-Aib-His-N-Me-D-2Nal-D-Phe-Lys-NH₂

H-Aib-His-D-2Nal-D-Phe-N-Me-Lys-NH₂

(3-Aminomethylthienyl-2-carbonyl)-D-2Nal-D-Phe-Lys-NH₂

H-Aib-His-D-2Nal-N-Me-D-Phe-Lys-NH₂

H-Aib-His-D-2Nal-D-Phe-Lys-N(Me)₂

(3R)-Piperidinecarbonyl-D-2Nal-D-Phe-Lys-NH₂

(3S)-Piperidinecarbonyl-D-2Nal-D-Phe-Lys-NH₂

(3-Aminomethylbenzoyl)-D-lNal-D-Phe-Lys-NH₂

H-Aib-His-D-2Nal-D-Trp-Lys-NH₂

(Furfuryl)-Aib-His-D-2Nal-D-Phe-Lys-NH₂

(2-Pyridylmethyl)-Aib-His-D-2Nal-D-Phe-Lys-NH₂

H-Aib-(3-aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂

H-Aib-3Pyal-D-2Nal-D-Phe-Lys-NH₂

(3S)-Piperidinecarbonyl-D-2Nal-D-Phe-Lys-NH₂

(3R)-Piperidinecarbonyl-D-2Nal-D-Phe-Lys-NH₂

(2-(H-Aib-His-D-2Nal-NH)ethyl)benzene

N,N-di(2R-Hydroxypropyl)-(3-aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂

(2R-Hydroxypropyl)-Aib-His-D-2Nal-D-Phe-Lys-NH₂

(3-Aminomethylbenzoyl)-D-2Nal-D-Pheψ(CH₂ NH)Lys-NH₂

(3-Aminomethylbenzoyl)-N-Me-D-2Nal-D-Phe-Lys-NH₂

(3-Aminomethylbenzoyl)-D-2Nal-D-Phe-N-Me-Lys-NH₂

H-D-Thr-His-D-2Nal-D-Phe-Lys-NH₂

H-Aib-His-D-2Nal-N-(phenethyl)-Gly-Lys-NH₂

(3-Aminomethylbenzoyl)-D-2Nal-N-(phenethyl)-Gly-Lys-NH₂

H-Hyp-His-D-2Nal-D-Phe-Lys-NH₂

H-Aib-His-N-Me-D-2Nal-N-(phenethyl)-Gly-Lys-NH₂

H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-Lys-NH₂

H-Aib-His-D-2Nal-D-Pheψ(CH₂ N(Me))Lys-NH₂

3-(H-Aib-His-D-2Nal-N-Me-D-Phe-NH)morpholinopropane

2-(H-Aib-His-D-2Nal-N-Me-D-Phe-NH)-( 1-methyl-2-pyrrolidinyl)ethane

(3R)-Piperidinecarbonyl-N-Me-D-2Nal-N-Me-D-Phe-Lys-NH₂

3-((Aminomethylbenzoyl)-D-2Nal-N-Me-D-Phe-NH)morpholinopropane

2-(H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NH)-(1-methyl-2-pyrrolidinyl)ethane

2-(3R)-Piperidinecarbonyl-N-Me-D-2Nal-N-Me-D-Phe-NH)-(1-methyl-2-pyrrolidinyl)ethane

2-(3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me-D-Phe-NH)-(1-methyl-2-pyrrolidinyl)ethane

3-(H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NH)morpholinopropane

3-((3R)-Piperidinecarbonyl-N-Me-D-2Nal-N-Me-D-Phe-NH)morpholinopropane

3-((3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me-D-Phe-NH)morpholinopropane

H-Aib-His-D-2Nal-N-Me-D-Phe-Hyp-NH₂

2-((3-Aminomethylbenzoyl)-D-2Nal-N-Me-D-Phe-NH)-(1-methyl-2-pyrrolidinyl)ethane

2-((3R)Piperidinecarbonyl-D-2NaI-N-Me-D-Phe-NH)-(1-methyl-2-pyrrolidinyl)ethane

Abbreviations:

D-2Nal=D-2-naphthylalanine

5Apent=5-aminopentanoic acid

3Pyal=3-pyridylalanine

Aib=H-amino-isobutyric acid

5 Thial=cthienylalanine

hp he homo-phenylalanine

N-Bzl-Gly=N-benyiglycine

4 F=4-fluoro

4-OMe=4-methoxy

10 Orn=ornithine

Dab=2,4-diaminobutyric acid

Hyp=hydroxyproline

Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

Compounds of formula I may be prepared by conventional methods ofsolution or solid phase peptide synthesis. For instance, solid phasesynthesis may be carried out substantially as described by Stewart andYoung, Solid Phase Pepatide Sythesis, 2nd. Ed., Rockford, Ill. USA,1976. Solution peptide synthesis may for instance be carried outsubstantially as described by Bodansky et al., Peptide Synthesis, 2nd.Ed., New York, N.Y., USA, 1976.

Aminomethylene as a substitution of an amide bond may be introducedaccording to the method described by Y. Sasaki and D. H. Coy, Peptides8(1), 1987, pp.119-121. Peptide derivatives containing a mono- ordi-hexapyranose derivatised amino group may be prepared by an Amadorirearrangement substantially be the method described by R. Albert et al.,Life Sciences 53 1993, pp.517-525. Examples of suitable mono- ordi-hexapyranoses are glucose, galactose, maltose, lactose or cellobiose.Derivatives used as starting materials in the synthesis may either beobtained commercially and, when required, provided with suitableprotecting groups, or starting materials used to prepare the "A" moietyin general formula I may b e prepare d by well-known methods andoptionally protected in a manner known per se.

Pharmaceutically acceptable acid addition salts of compounds of formulaI include those prepared by reacting the peptide with an inorganic ororganic acid such as hydrochloric, hydrobromic, sulfuric, acetic,phosphoric, lactic, maleic, phthalic, citric, glutaric, gluconic,methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic,trifluoracetic, sulfamic and fumaric acid.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising, as an active ingredient, a compound of thegeneral formula I or a pharmaceutically acceptable salt thereof togetherwith a pharmaceutically acceptable carrier or diluent. Pharmaceuticalcompositions containing a compound of the present invention may beprepared by conventional techniques, e.g. as described in Remington'sPharmaceutical Sciences, 1985. The compositions may appear inconventional forms, for example capsules, tablets, aerosols, solutions,suspensions or topical applications.

The pharmaceutical carrier or diluent employed may be a conventionalsolid or liquid carrier. Examples of solid carriers are lactose, terraalba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid or lower alkyl ethers of cellulose.Examples of liquid carriers are syrup, peanut oil, olive oil,phospholipids, fatty acids, fatty acid amines, polyoxyethylene andwater.

Similarly, the carrier or diluent may include any sustained releasematerial known in the art, such as glyceryl monostearate or glyceryldistearate, alone or mixed with a wax.

If a solid carrier is used for oral administration, the preparation maybe tabletted, placed in a hard gelatin capsule in powder or pellet formor it can be in the form of a troche or lozenge. The amount of solidcarrier will vary widely but will usually be from about 25 mg to about 1g.

A typical tablet which may be prepared by conventional tablettingtechniques may contain:

    ______________________________________                                        Core:                                                                         Active compound (as free compound or salt thereof)                                                       100    mg                                          Colloidal silicon dioxide (Aerosil)                                                                      1.5    mg                                          Cellulose, microcryst. (Avicel)                                                                          70     mg                                          Modified cellulose gum (Ac--Di--Sol)                                                                     7.5    mg                                          Magnesium stearate                                                            Coating:                                                                      HPMC approx.               9      mg                                          *Mywacett 9-40 T approx.   0.9    mg                                          ______________________________________                                         *Acylated monoglyceride used as plasticizer for film coating.            

If a liquid carrier is used, the preparation may be in the form of asyrup, emulsion, soft gelatin capsule or sterile injectable liquid suchas an aqueous or non-aqueous liquid suspension or solution.

For nasal administration, the preparation may contain a compound offormula I dissolved or suspended in a liquid carrier, in particular anaqueous carrier, for aerosol application. The carrier may containadditives such as solubilizing agents, e.g. propylene glycol,surfactants such as bile acid salts or polyoxyethylene higher alcoholethers, absorption enhancers such as lecithin (phosphatidylcholine) orcyclodextrin, or preservatives such as parabenes.

Generally, the compounds of the present invention are dispensed in unitdosage form comprising 0.0001-100 mg of active ingredient together witha pharmaceutically acceptable carrier per unit dosage.

The dosage of the compounds according to this invention is suitably1-500 mg/day, e.g. about 100 mg per dose, when administered to patients,e.g. humans, as a drug.

It has been demonstrated that compounds of the general formula I possessthe ability to release endogenous growth hormone in vivo. The compoundsmay therefore be used in the treatment of conditions which requireincreased plasma growth hormone levels such as in growth hormonedeficient humans or in elderly patients or livestock.

Thus, in a particular aspect, the present invention relates to apharmaceutical composition for stimulating the release of growth hormonefrom the pituitary, the composition comprising, as an active ingredient,a compound of the general formula I or a pharmaceutically acceptablesalt thereof together with a pharmaceutically acceptable carrier ordiluent.

In a further aspect, the present invention relates to a method ofstimulating the release of growth hormone from the pituitary, the methodcomprising administering to a subject in need thereof an effectiveamount of a compound of the general formula I or a pharmaceuticallyacceptable salt thereof.

In a still further aspect, the present invention relates to the use of acompound of the general formula I or a pharmaceutically acceptable saltthereof for the preparation of a medicament for stimulating the releaseof growth hormone from the pituitary.

To those skilled in the art, it is well known that the current andpotential uses of growth hormone in humans are varied and multitudinous.It is anticipated that, compounds of formula I can be administered forpurposes of stimulating release of growth hormone from the pituitary andwould then have similar effects or uses as growth hormone itself. Theuses of growth hormone may be summarized as follows: stimulation ofgrowth hormone release in the elderly; prevention of catabolic sideeffects of glucocorticoids, treatment of osteoporosis, stimulation ofthe immune system, acceleration of wound healing, accelerating bonefracture repair, treatment of growth retardation, treating renal failureor insufficiency resulting from growth retardation, treatment ofphysiological short stature including growth hormone deficient childrenand short stature associated with chronic illness, treatment of obesityand growth retardation associated with obesity, treating growthretardation associated with the Prader-Willi syndrome and Turner'ssyndrome; accelerating the recovery and reducing hospitalization of burnpatients; treatment of intrauterine growth retardation, skeletaldysplasia, hypercortisolism and Cushing's syndrome; induction ofpulsatile growth hormone release; replacement of growth hormone instressed patients, treatment of osteochondrodysplasias, Noonan'ssyndrome, schizophrenia, depressions, Alzheimer's disease, delayed woundhealing and psychosocial deprivation, treatment of pulmonary dysfunctionand ventilator dependency, attenuation of protein catabolic responsesafter major surgery, reducing cachexia and protein loss due to chronicillness such as cancer or AIDS; treatment of hyperinsulinemia includingnesidioblastosis, adjuvant treatment for ovulation induction; tostimulate thymic development and prevent the age-related decline ofthymic function, treatment of immunosuppressed patients, improvement inmuscle strength, mobility, maintenance of skin thickness, metabolichomeostasis, renal homeostasis in the frail elderly, stimulation ofosteoblasts, bone remodelling and cartilage growth, stimulation of theimmune system in companion animals and treatment of disorder of aging incompanion animals, growth promoter in livestock and stimulation of woolgrowth in sheep.

For the above indications the dosage may vary depending on the compoundof formula I employed, on the mode of administration and on the therapydesired. However, generally dosage levels between 0.0001 and 100 mg/kgbody weight per day may be administered to patients and animals toobtain effective release of endogenous growth hormone. Usually, dosageforms suitable for oral or nasal administration comprise from about0.0001 mg to about 100 mg, preferably from about 0.001 mg to about 50 mgof the compounds of formula I admixed with a pharmaceutically acceptablecarrier or diluent.

The compounds of formula I may be administered in pharmaceuticallyacceptable acid addition salt form or, where appropriate, as a alkalimetal or alkaline earth metal or lower alkylammonium salt. Such saltforms are believed to exhibit approximately the same order of activityas the free base forms.

Optionally, the pharmaceutical composition of the invention may comprisea compound of formula I combined with one or more compounds exhibiting adifferent activity, e.g., an antibiotic or other pharmacologicallyactive material. This might be another secretagogue, such as GHRP (1 or6) or GHRH or an analogue thereof, growth hormone or an analogue thereofor a somatomedin such as IGF-1 or IGF-2.

The route of administration may be any route which effectivelytransports the active compound to the appropriate or desired site ofaction, such as oral, nasal or parenteral, the oral route beingpreferred.

Apart from the pharmaceutical use of the compounds of formula I, theymay be useful in vitro tools for investigating the regulation of growthhormone release.

The compounds of formula I may also be useful in vivo tools forevaluating the growth hormone releasing capability of the pituitary. Forexample, serum samples taken before and after administration of thesecompounds to humans can be assayed for growth hormone. Comparison of thegrowth hormone in each serum sample would directly determine the abilityof the patient's pituitary to release growth hormone.

Compounds of formula I may be administered to commercially importantanimals to increase their rate and extent of growth, and to increasemilk production.

Pharmacological Methods

Compounds of formula I may be evaluated in vitro for their efficacy andpotency to release growth hormone in primary rat somatotrophs.

Rat primary somatotrophs may be prepared essentially as describedpreviously (Chen t al., Endocrinology 1991, 129, 3337-3342 and Chen etal., Endocrinology 1989, 124, 2791-2798). Briefly, rats are killed bydecapitation. The pituitary is quickly removed. H pituitaries aredigested with 0.2% collagenase and 0.2% hyalurinidase in Hanks balancedsalt solution. The cells are resuspended in Dulbecco's Modified Eagle'smedium containing 0.37% NaHCO3, 10% horse serum, 2.5% fetal calf serum,1% nonessential amino acids, 1% glutamine and 1% penicillin/streptomycinand adjusted to 1.5×105 cells/ml. One ml of this suspension is placed ineach well of 24-well trays and left for 2-3 days before releaseexperiments are performed.

On day one of the experiments, cells are washed twice with the abovemedium containing 25 mM HEPES, pH 7.4. Growth hormone release initiatedby addition of medium containing 25 mM HEPES and test compound.Incubation is carried out for 15 minutes at 37° C. After incubationgrowth hormone released to the medium is measured by a standard RIA.

Compounds of formula I may be evaluated for their in vivo effects ongrowth hormone release in pentobarbital anaesthetized female rats asdescribed previously (Bercu et al. Endocrinology 1991, 129, 2592-2598).Briefly, adult male Sprague-Dawley rats are anesthetized withpentobarbital 50 mg/kg ip. After the rats had been fully anaesthesizedthe rats are implanted with a tracheal cannula and catheters in thecarotid artery and the jugular vein. After a 15 minute recovery, a bloodsample is taken at time 0. The pituitary secretagogues are administerediv and artery blood samples are put on ice for 15 minutes and thencentrifuged for 2 minutes at 12,000 ×g. The serum is decanted and amountof growth hormone determined using a standard RIA.

The invention is further illustrated in the following examples which arenot in any way intended to limit the scope of the invention as claimed.

The abbreviations used throughout this description and examples indicatethe following structures:

Abbreviations for non-natural amino acid residues: ##STR14##

Abbreviations used for peptide bond substitutions: ##STR15##

Abbreviations used for protecting groups: ##STR16##

The compounds prepared in the following examples were all isolated asthe trifluoroacetic acid (TFA) salts.

EXAMPLE 1 H-Ala-His-D-2Nal-D-Trp-Lys-NH₂

The title peptide was synthesized according to the Fmoc strategy on anApplied Biosystems 431A peptide synthesizer in 0.22 mmol scale using themanufacturer supplied FastMoc UV protocols which employ HBTU(2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluroniumhexafluorophosphate) mediated couplings in NMP (N-methyl pyrrolidone)and UV monitoring of the deprotection of the Fmoc protection group. Thestarting resin used for the synthesis was 559 mg 4-(1, 2',4'-dimethoxyphenyl)-(Fmoc-amino)methyl)-phenoxy resin (Novabiochem, BadSoden, Germany. cat. #: 01-64-0013) with a substitution capacity of 0.39mmol/g. The protected amino acid derivatives used were Fmoc-Lys(Boc)-OH,Fmoc-D-Trp-OH, Fmoc-2Nal-OH, Fmoc-His(Trt) and Fmoc-Ala-OH.

The peptide was cleaved from 434 mg of the peptide resin by stirring for180 min at room temperature with a mixture of 4 ml TFA (trifluoroaceticacid), 300 mg phenol, 100 μl ethanedithiol, 200 μl thioanisole and 200μl H₂ O. The cleavage mixture was filtered and the filtrate wasconcentrated to 1 ml by a stream of nitrogen. The crude peptide wasprecipitated from this oil with 45 ml diethyl ether and washed 3 timeswith 50 ml diethyl ether.

The crude peptide was dryed and purified by semipreparative HPLC on a 20mm x 250 mm column packed with 7μC-18 silica which was preequilibratedwith 15% CH₃ CN in 0.05M (NH₄)₂ SO₄, which was adjusted to pH 2.5 with4M H₂ SO₄. The crude peptide was dissolved in 2 ml 70% CH₃ CN / 0.1% TFAin H₂ O and diluted to 100 ml with H₂ O.

This solution was divided into two equal portions and each of them wereinjected on the column in two separate runs. The column was eluted witha gradient of 15%-25% CH₃ CN in 0.05M (NH₂),SO₄, pH 2.5 at 10 ml/minduring 47 min at 40° C. The peptide containing fractions were collected,diluted with 3 volumes of H M0 and applied to a SEP-PAK® C18 silicacartridge (Waters part. #:51910) which was equilibrated with 0.1% TFA.The peptide was eluted from the SEP-PAK® C18 silica 5 cartridge with 70%CH₃ CN 0.1% T2A and isolated from the eluate by lyophilisation afterdilution with water. A yield of 19.0 mg was obtained.

The final product obtained was characterised by analytical RP-HPLC(retention time) and by Plasma desorption mass spectrometry (molecularmass). Mass spectrometry agreed with the expected structure within theexperimental error of the method (mass spectrometry±0.9 amu).

The RP-HPLC analysis was performed using UV detection at 214 nm and aVydac 2 18TP54 4.6 mm×250 mm 5μC-18 silica column (The SeparationsGroup, Hesperia) which was eluted at 1ml/min at 42° C. Two differentelution conditions were used:

A1: The column was equilibrated with 5% CH₃ CN in a buffer consisting of0.1M (NH₂),SO₄ 1 Which was adjusted to pH 2.5 with 4 M H₂ SO₄ and elutedby a gradient of 5% to 60% CH₃ CN in the same buffer during 50 min.

B1: The column was equilibrated with 5% CH₃ CN i 0.1% T iA i H w t an deluted by a gradient of 5% CH₃ CN/0.1% TFA/H₂ O to 60% CH₃ CN/0.1%TFA/H₂ O during 50 min.

The retention time using elution conditions Al and Bi was found to be17.88 m i and 20.15 min, respectively.

EXAMPLE 2 H-Ala-His-D-2Nal-D-Phe-Lys-OH

The title peptide was synthesized using a procedure similar to the onedescribed in example 1. with the exception that 450 mgFmoc-Lys(Boc)-Wang resin (Novabiochem, Bad Soden, Germany. cat. #04-12-2014) with a substitution capacity of 0.49 mmol/g was used as thestarting resin. Upon cleavage of 560 mg peptide resin and purificationof 1/2 of the resulting crude product as described in example 1. a yieldof 25.9 mg was obtained.

The final product was characterised as described in Example 1. Theretention time using elution conditions Al and B i was found to be 18.30min and 20.15 min, respectively.

EXAMPLE 3 5-(H-Ala-His-D-2Nal-D-Phe-NH)aminopentane

The peptide resin H-Ala-His(Trt)-D-2Nal-D-Phe- Sasrin resin wassynthesized using a procedure similar to the one described in Example 1.with the exception that 262 mg Sasrin resin (2-Methoxy-4-alkoxybenzylalcohol resin) (Bachem, Bubendorf, Switzerland cat. # D-1295) with asubstitution capacity of 0.96 mmol/g was used and the protocol used forcoupling of the first amino acid residue to the resin was a4-dimethylaminopyridine catalysed coupling of the preformed symmetricalanhydride followed by capping of residual --OH groups on the resin withbenzoic anhydride.

The partially protected peptide5-(H-Ala-His(Trt)-D-2Nal-D-Phe-NH)aminopentane was cleaved from 56 mg ofthe H-Ala-His(Trt)-D-2Nal-D-Phe- Sasrin resin by stirring for 20 h atroom temperature with 0.5 ml of 1,5-diaminopentane. The spent resin wasfiltered off and extracted with 1 ml DMF. The combined filtrate andextract was slowly added to a mixture of 2.5 ml CH₃ CN and 10 ml 1Mhydrochloric acid under stirring and after dilution to 50 ml with 25%CH₃ CN the mixture was left at 4 ° C. for 100 h (for cleavage of thetrityl protection on the histidine). The mixture was then diluted to 200ml with H₂ O and filtered.

The crude peptide was purified by semipreparative HPLC by directinjection on the column of 1/2 of this filtrate using a proceduresimilar to that described in example 1. The yield was 4.5 mg.

The final product was characterised as described in Example 1. Theretention time using elution conditions A 1and B 1was found to be 18.43min and 20.75 min, respectively.

EXAMPLE 4 (2S)-(H-Ala-His-D-2Nal-D-Phe-NH)-6-aminohexanol

The peptide resin H-Ala-His(Trt)-D-2Nal-D-Phe-Lys(Boc)-Sasrin resin wassynthesized using a procedure similar to the one described in Example 3from a Sasrin resin with a substitution capacity of 0.96 mmol/g.

The partially protected peptide(2S)-(H-Ala-His(Trt)-D-2Nal-D-Phe-NH)-6-aminohexanol was cleaved from200 mg of the H-Ala-His(Trt)-D-2Nal-D-Phe-Lys(Boc)-Sasrin resin bystirring the peptide resin for 20 h at room temperature with a mixtureof 1.2 ml THF (tetrahydrofuran) 0.2 ml ethanol, 23 mg LiBr and 10 mgNaBH₄. Then 200 μl H₂ O, 200 μl acetic acid and 4 ml ethanol was added.The resin beads were removed by filtration and the filtrate was dilutedwith 50 ml H₂ O and lyophilized. The resulting powder was subjected toTFA cleavage and purification as described in example 1. Thepurification had to be repeated in order to obtain a sufficiently pureproduct. A yield of 5.8 mg was obtained.

The final product was characterised as described in Example 1. Theretention time using elution conditions A1 and B1 was found to be 17.82min and 20.02 min respectively.

EXAMPLE 5

H-Ala-Hisψ(CH₂ NH)D-2Nal-D-Phe-Lys-NH₂

The peptide resin H-D-2Nal-D-Phe-Lys-(Boc)Resin was synthesized using aprocedure similar to the one described in Example 1 starting from 556 mg4-((2',4'-dimethoxyphenyl)-(Fmoc-amino)methyl)-phenoxy resin resin(Novabiochem AG Switzerland, cat. #: 01-64-0013) having a substitutioncapacity of 0.34 mmol/g . The following protected amino acid derivativeswere used: Fmoc-Lys(Boc)-OH, Fmoc-D- Phe-OH and Fmoc-2Nal-OH.

The -CH₂ NH- peptide bond isostere was introduced according to Sasaki,Y. and Coy, D. H., PEPTIDES 8(1) 119-121. 1987.

Fmoc-His(Trt)-aldehyde was prepared from 820 mg of the corresponding N,O-dimethyl hydroxamate according to Fehrentz, J. -A. and Castro. B.,SYNTHESIS 676-678. 1983. The crude aldehyde was dissolved in 8 ml DMFand divided into two portions. The first portion was added to a stirredslurry of 610 mg of H-D-2Nal-D-Phe-Lys-Resin in 10 ml 1% acetic acid inDMF at room temperature. Then 57 mg NaCNBH₃ (85% pure) dissolved in 1 mlDMF was added and stirring was continued for 60 min. After this thepeptide resin was isolated by filtration and washed with 1% acetic acidin DMF. The peptide resin was again suspended in 10 ml 1% acetic acid inDMF and the second portion of the Fmoc-His(Trt)-aldehyde was added.Again 57 mg NaCNBH₃ (85% pure) dissolved in 1 ml DMF was added at roomtemperature and the mixture was stirred for 18 h.

After this reductive alkylation step, the peptide resin was isolated byfiltration and washed with 1% acetic acid in DMF and the chainelongation was completed using the peptide synthesizer according to theabove described procedures using the protected amino acid derivativeFmoc-Ala-OH.

The peptide was cleaved from 550 mg of the peptide resin and the crudepeptide was purified by semipreparative HPLC using a procedure similarto that described in Example 1. A yield of 11.3 mg was obtained.

The final product was characterised as described in Example 1. Theretention time using elution conditions Al and B i was found to be 13.35min and 17.38 min respectively.

EXAMPLE 6

(n-Propyl)-His-D-2Nal-D-Phe-Lys-NH₂ and (n-propyl)₂-His-D-2-Nal-D-Phe-Lys-NH₂

The peptide resin H-His(Trt)-D-2Nal-D-Phe-Lys(Boc)-Resin was synthesizedusing a procedure similar to the one described in Example 1 startingfrom 4-((2',4'-dimethoxyphenyl)-(Fmoc-amino)methyl)-phenoxy resin resin(Novabiochem AG Switzerland, cat. #: 01-64-0013 ) having a substitutioncapacity of 0.34 mmol/g. The protected amino acid derivatives used wereFmoc-Lys(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-2Nal-OH and Fmoc-His(Trt)-OH.

13 μl n-propanal was added to a stirred slurry of 150 mg ofH-His(Trt)-D-2Nal-D-Phe-Lys-Resin in 3.3 ml 1% acetic acid in DMF atroom temperature. Then 16.8 mg NaCNBH₃ (85% pure) dissolved in 0.5 mlDMF was added and stirring was continued for 6 h. After this reductivealkylation step, the peptide resin was isolated and washed on a filterfunnel with DMF and CH₂ Cl₂ and then dried in vac.

The two resulting peptide resins were mixed and a mixture ofunalkylated, mono-n propyl and di-n-propyl peptide was cleaved from theresulting 300 mg peptide resin. The peptides were separated and purifiedby semipreparative HPLC using a procedure similar to that described inexample 1. A yield of 6.54 mg of n-propyl-His-D-2Nal-D-Phe-Lys-NH₂ and5.59 mg of (n-propyl)₂ -His-D-2Nal-D-Phe-Lys-NH₂ was obtained.

The final products were characterised as described in example 1. For(n-propyl)-His-D-2Nal-D-Phe-Lys-NH₂ the retention time using elutionconditions A1 and B1 was found to be 16.45 min and 19.92 min,respectively.

EXAMPLE 7

H-Ala-Tic-D-2Nal-D-Phe-Lys-NH₂

Starting from 620 mg 4-methyl BHA resin (Bissendorf Biochemicals,Hannover, Germany. cat. #: RMIS50) having a substitution capacity of0.72 mmol/g the peptide was synthesized according to the Boc strategy onan Applied Biosystems 430A peptide synthesizer using the manufacturersupplied 0.5 mmol scale single coupling protocols which employ singlecouplings with preformed symmetrical anhydrides in DMF. The protocolswere adjusted to 60 min coupling time. A double coupling with 2×60 mincoupling time was used for the N-terminal Ala. The protected amino acidderivatives used for the synthesis was Boc-Lys(2-chloro-Z)-OH,Boc-D-Phe-OH, Boc-D-2Nal-OH, Boc-Tic-OH and Boc-Ala-OH.

The peptide was cleaved from 486 mg of the peptide resin by stirring itfor 75 min at 0° C. with a mixture of 4.5 ml HF and 500, μl m-cresol.The HF was evaporated at 0° C. by a stream of nitrogen. The peptide wasprecipitated from the remaining oil together with the spend resin with50 ml diethyl ether and washed 2 times with 50 ml diethyl ether. Afterdrying the peptide was extracted precipitate with 10 ml H₂ O containing4 drops of acetic acid. The extract was diluted to 100 ml H₂ O.

The crude peptide was purified from 2×18 ml of the diluted extract bytwo runs of semipreparative HPLC using a procedure similar to thatdescribed in example 1. The yield was 17.3 mg.

The final product was characterised as described in example 1.

RP-HPLC analysis using conditions A1 and B1 gave the retention times27.37 min and 29.50 min respectively.

EXAMPLE 8

(2R)-(H-Ala-His-D-2Nal-NH)-3-phenylpropylamine

The Fmoc group was removed by treatment with 20% piperidine in NMP for20 min from 580 mg4-((2',4'-dimethoxyphenyl)-(Fmoc-amino)methyl)-phenoxy resin resin(Novabiochem AG Switzerland, cat. #: 01-64-0013) having a substitutioncapacity of 0.43 mmol / g. The resin was washed with DMF and with CH₂Cl₂ and alkylated with Fmoc-D-Phe-aldehyde using a reductive alkylationprocedure described in example 5.

After this, the peptide resin was isolated by filtration and washed with1% acetic acid in DMF on a filter funnel and the chain elongation wascompleted using the peptide synthesizer as described in example 1. usingthe protected amino acid derivatives Fmoc-D-2Nal-OH, Fmoc-His(Trt)-OHand Fmoc-Ala-OH.

The peptide was cleaved from 301 mg of the peptide resin and the crudepeptide was purified by semipreparative HPLC using a procedure similarto that described in example 1. A yield of 23.92 mg was obtained.

The final product was characterised as described in Example 1. Theretention time using elution conditions A1 and B1 was found to be 19.33min and 21.77 min respectively.

    __________________________________________________________________________                                 RP-                                                                       Pre-                                                                              HPLC                                                                              RP-                                                                   pared                                                                             reten-                                                                            HPLC                                                                  using a                                                                           tion                                                                              reten-                                                                pro-                                                                              time                                                                              tion                                                                  cedure                                                                            for time                                                                  similar                                                                           condi-                                                                            for                                                                   to  tion                                                                              condi-                                                                Exam-                                                                             A1  tion B1                                      Ex.                                                                             Peptide                ple (Ex. 1)                                                                           (Ex. 1)                                      __________________________________________________________________________     9                                                                              H--Ala--His--D--Phe--D--Phe--Lys--NH.sub.2                                                           1   12.53                                                                             15.43                                        10                                                                              H--Ala--Phe--D-2Nal-D--Phe--Lys--NH.sub.2                                                            1   28.13                                                                             29.62                                        11                                                                              H--His--D-2Nal-D--Phe--Lys--NH.sub.2                                                                 1   18.02                                                                             20.15                                        12                                                                              H--Ala--His--D-2Nal-D--Phe--Phe--NH.sub.2                                                            1   28.48                                                                             29.48                                        13                                                                              H--Ala--His--D-2Nal-D--Phe--D--Phe--NH.sub.2                                                         1   26.65                                                                             27.75                                        14                                                                              H--Ala--His--D-2Nal-Phe--Lys--NH.sub.2                                                               1   21.85                                                                             23.12                                        15                                                                              (3-(4-Imidazolyl)propionyl)-D-2Nal-D--Phe--                                                          1   20.7                                               Lys--NH.sub.2                                                               16                                                                              (Propionyl)-His--D-2Nal-D--Phe--Lys--NH.sub.2                                                        1   22.2                                                                              23.77                                        17                                                                              H--Ala--His--D-2Nal-D--Phe--NH.sub.2                                                                 1   21.7                                                                              23.08                                        18                                                                              (H--Ala--His--D-2Nal-D--Phe--NH)hexane                                                               3   34.11                                                                             35.78                                        19                                                                              H--Ala--His--D--Trp--D--Phe--Lys--NH.sub.2                                                           1   14.52                                                                             16.7                                         20                                                                              H--Ala--Ala--D-2Nal-D--Phe--Lys--NH.sub.2                                                            1   21.97                                                                             23.23                                        21                                                                              ((Propionyl)-His--D-2Nal-D--Phe--                                                                    3   37.17                                                                             39.47                                          NH)hexane                                                                   22                                                                              6-(H--Ala--His--D-2Nal-D--Phe--                                                                      3   19.3                                                                              21.57                                          NH)hexylamine                                                               23                                                                              H--D-2Nal-D--Phe--Lys--NH.sub.2                                                                      1   15.88                                                                             18.25                                        24                                                                              (5-Aminopentanoyl)--His--D-2Nal-D--Phe--                                                             1   17.8                                                                              19.98                                          Lys--NH.sub.2                                                               25                                                                              H--D--Lys--D-2Nal-D--Phe--Lys--NH.sub.2                                                              1   17.07                                                                             19.62                                        26                                                                              H--Ala--His--D-2Nal-D--Tic--Lys--NH.sub.2                                                            1   19.12                                                                             20.78                                        27                                                                              H--D--Lys--Phe-2Nal-D--His--D--Ala--NH.sub.2                                                         1   18.15                                                                             20.48                                        28                                                                              (5--Aminopentanoyl)--D-2Nal-D--Phe--Lys--                                                            1   20.67                                                                             22.45                                          NH.sub.2                                                                    29                                                                              H--D--Ala--D-2Nal-D--Phe--Lys--NH.sub.2                                                              1   19.57                                                                             21.53                                        30                                                                              (Propionyl)--D-2Nal-D--Phe--Lys--NH.sub.2                                                            1   26.7                                                                              27.92                                        31                                                                              H--D--Ala--His--D-2Nal-D--Phe--Lys--NH.sub.2                                                         1   17.83                                                                             20.3                                         32                                                                              H--Ala-3-Pyal--D-2Nal-D--Phe--Lys--NH.sub.2                                                          1   18.15                                                                             20.18                                        33                                                                              (n-Butyl)-Ala--His--D-2Nal-D--Phe--Lys--NH.sub.2                                                     6   19.55                                                                             --                                           34                                                                              (n-Octyl)-Ala--His--D-2Nal-D--Phe--Lys--NH.sub.2                                                     6   27.88                                                                             24.52                                        35                                                                              H--Ala--His--D-2Nal-D--Pheψ(CH.sub.2 NH)Lys--                                                    5   17.97                                                                             20.83                                          NH.sub.2                                                                    36                                                                              (3-Aminomethylbenzoyl)-D-2Nal-D--Phe--                                                               1   22.42                                                                             24.13                                          Lys--NH.sub.2                                                               37                                                                              (3-Aminophenylacetyl)-D-2Nal-D--Phe--Lys--                                                           1   23.62                                                                             23.97                                          NH.sub.2                                                                    38                                                                              ((4-Imidazolyl)acetyl)-D-2Nal-D--Phe--Lys--                                                          1   20.42                                                                             22.22                                          NH.sub.2                                                                    39                                                                              (3-(4-Imidazolyl)acryloyl)-D-2Nal-D--Phe--                                                           1   21.25                                                                             23.32                                          Lys--NH.sub.2                                                               40                                                                              (4-Aminophenylacetyl)-D-2Nal-D--Phe--Lys--                                                           1   22.45                                                                             --                                             NH.sub.2                                                                    41                                                                              (Trans-4-aminomethylcyclohexanoyl)-D-                                                                1   22.07                                                                             23.67                                          2Nal-D--Phe--Lys--NH.sub.2                                                  42                                                                              2-(H--Ala--His--D-2Nal-D--Phe--NH)-                                                                  3   18.6                                                                              20.25                                          ethylamine                                                                  43                                                                              (2-(H--Ala--His--D-2Nal-D--Phe--                                                                     3   30.18                                                                             32.18                                          NH)ethyl)benzene                                                            44                                                                              4-((H--Ala--His--D-2Nal-D--Phe--                                                                     3   19.63                                                                             21.55                                          NH)methyl)benzylamine                                                       45                                                                              (2R)-(H--Ala--His--D-2Nal-NH)-3-                                                                     4   21.55                                                                             23.57                                          phenylpropylamine                                                           46                                                                              2-(H--Ala--His--D-2Nal-NH)ethylamine                                                                 3   25.52                                                                             --                                           47                                                                              H--D--Phe--Ala--D-2Nal-D--Phe--Lys--NH.sub.2                                                         1   27.82                                                                             29.43                                        48                                                                              H--Ala--D--Phe--D-2Nal-D--Phe--Lys--NH.sub.2                                                         1   25.62                                                                             27.22                                        49                                                                              H--Ala--(2-aminobenzoyl)-D-2Nal-D--Phe--                                                             7   26.42                                                                             24.93                                          Lys--NH.sub.2                                                               50                                                                              H--Aib--His--D-2Nal-D--Phe--Lys--NH.sub.2                                                            1   17.42                                                                             20.13                                        51                                                                              H--Ala--His--D-1Nal-D--Phe--Lys--NH.sub.2                                                            1   17.55                                                                             19.8                                         52                                                                              H--Tyr--Ala--His--D-2Nal-D--Phe--Lys--NH.sub.2                                                       1   19.9                                                                              21.22                                        53                                                                              (Piperidine-4-carbonyl)-D-2Nal-D--Phe--Lys--                                                         1   20.4                                                                              22.32                                          NH.sub.2                                                                    54                                                                              H--Ala--Phe(4-NH.sub.2)--D-2Nal-D--Phe--Lys--NH.sub.2                                                1   19.20                                                                             --                                           __________________________________________________________________________

EXAMPLE 55

((Propionyl)-D-2Nal-D-Phe-NH)hexane

The peptide resin (Propionyl)-D-2Nal-D-Phe-Sasrin resin was synthesizedusing a procedure similar to the one described in Example 3 from aSasrin resin with a substitution capacity of 0.96 mmol/g.

105 mgof the resulting peptide resin was subjected to ammonolysis.

The peptide ((Propionyl)-D-2Nal-D-Phe-NH)hexane was cleaved from 105 mgof the Propionyl-D-2Nal-D-Phe-Sasrin resin by stirring for 20 h at roomtemperature with 1 ml of n-hexylamine. The spent resin was filtered offand extracted with 1 ml DMF. The combined filtrate and extract wasslowly added to 8 ml 1 M hydrochloric acid under stirring. The resultingprecipitate was redissolved by addition of approx. 70 ml CH₃ CN and wasthen reprecipitated by addition of 20 ml H₂ O. The precipitate wasfiltered off, washed with H₂ O and dryed. The yield was 12 mg.

This product was characterised as described in Example 1. with theexception that only one HPLC analysis using conditions similar to B1with the exception that a gradient of 0.1% TFA / H₂ O to 90% CH₃ CN /0.1% TFA / H₂ O during 50 min. was used. The retention time was found tobe 35.73 min.

EXAMPLE 56

(3-Methylaminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂

The peptide resin (3-Aminomethylbenzoyl)-D-2Nal-D-Phe-Lys(CI-Z)-MBHAresin was synthesized using a procedure similar to the one described inExample 7 from a MBHA resin with a substitution capacity of 0.72 mmol/g.

The resulting peptide resin was methylated according to Kaljuste, K. andUnden, A., Int. J. Peptide Protein Res. 42 118-124. 1993. 300 mg resinwas stirred for 2 h with a mixture of 8 ml DCM, 150 μl DIEA(diethyl-isopropylamine)and 39mg 4,4'-dimethoxydityl chloride (DOD-Cl)and then isolated by filtration and washed with DCM and with DMF. TheDOD protected peptide resin was then stirred with 18 ml of a 3.7%formaldehyde solution in DMF, 0.18 ml acetic acid and 180 mg NaCNBH₃ wasthen added and stirring was continued for 18 h. The resin was isolatedby filtration and washed with DMF and with DCM. The DOD protection wasthen removed by treatment with 2×3 ml DCM/TFA 1:1 for 5 min+30 minrespectively and the peptide resin was washed with DCM and then dried invacuum.

The resulting N-methylated peptide was cleaved from 370 mg of theresulting resin using a procedure similar to that described in example 7and it was purified and characterized as described in example 1. Theyield was 17.3 mg.

RP-HPLC analysis using conditions A1 and B1 gave the retention times of22.83 min and 24.60 min respectively.

EXAMPLE 57

(3-Dimethylaminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂

The peptide resin (3-Aminomethylbenzoyl)-D-2Nal-D-Phe-Lys(CI-Z)-MBHAresin was synthesized using a procedure similar to the one described inExample 7 from a MBHA resin with a substitution capacity of 0.72 mmol/g.

650 mg of the resulting peptide resin was then stirred with 20 ml 1%acetic acid in DMF and 45 μl of a 3.7% formaldehyde solution in DMF. 55mg NaCNBH₃ (85%) was then added and stirring was continued for 18 h. Theresin was isolated by filtration and washed with DMF, DCM/MeOH 6:4 andwith DCM.

The resulting mixture of N,N-di-methylated peptides was cleaved from 631mg of the resulting resin using a procedure similar to that described inexample 7 and it was purified and characterized as described inexample 1. The yield was 8.58 mg.

RP-HPLC analysis using conditions A1 and B1 gave the retention times of31.58 min and 33.00 min respectively.

EXAMPLE 58

(3-Amino-3-methylbutanoyl)-D-2Nal-D-Phe-Lys-NH₂

The peptide resin H-D-2Nal-D-Phe-Lys(Boc)-Rink resin was synthesizedusing a procedure similar to the one described in Example 1 from 2 g4-((2',4'-dimethoxyphenyl)-(Fmoc-amino)methyl)-phenoxy resin (Rinkresin) (Novabiochem, Bad Soden, Germany. cat. #: 01-64-0013) with asubstitution capacity of 0.39 mmol/g.

500 mg of the resulting peptide resin (0.15 mmol) was suspended in 4 mlDCM/MeOH 1:1. 42 μl triethylamine (0.3 mmol) was added and after coolingto 0° C., 31 mg (0.158 mmol) of2,2-dimethyl-4-oxo-azetidine-1-sulfonylchloride was added understirring. Stirring was continued 20 min at 0° C. and 90 min at roomtemperature. After washing the resin with DCM/MeOH 6:4 and drying invacuum, the crude peptide was cleaved from the resin and purified usingprocedures similar to those described in example 1. The yield was 41.81mg.

RP-HPLC analysis using conditions A1 and B1 gave the retention times of21.35 min and 22.95 min respectively.

EXAMPLE 59

(2S)-((3-Aminomethylbenzoyl)ψ(CH₂ NH)D-2Nal-D-Phe-NH)-6-aminohexanol

The peptide resin H-D-2Nal-D-Phe-Lys(Boc)- Sasrin resin was synthesizedusing a procedure similar to the one described in Example 3 from 980 mgSasrin resin with a substitution capacity of 0.87 mmol/g.

1.4 g of this resin was reductively alkylated with Boc-3-aminomethylbenzaldehyde and the peptide was cleaved from 1.0 g of the resultingresin and half of the crude product was purified the using proceduressimilar to those described in example 5. The yield was 18.46 mg.

RP-HPLC analysis using conditions A1 and B1 gave the retention times of14.78 min and 17.40 min respectively.

EXAMPLE 60

(2-Aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂

The peptide resin H-D-2Nal-D-Phe-Lys(Boc)-Rink resin was synthesizedusing a procedure similar to the one described in Example 1 from 2 g4-((2',4'-dimethoxyphenyl)-(Fmoc-amino)methyl)-phenoxy resin (Rinkresin) (Novabiochem, Bad Soden, Germany. cat. #: 01-64-0013) with asubstitution capacity of 0.39 mmol/g.

300 mg of the resulting peptide resin (0.096 mmol) was stirred 18 h in10 ml DMF with 54 mg phthaloyl-2-aminomethyl-benzoic acid (0.192 mmol),182 mg HBTU (0.480 mmol)and 164 μl DIEA (0.96 mmol).

After washing the resin with DMF, DCM/MeOH 6:4 and DCM and drying invacuum (Phthaloyl-2-aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂ was cleavedfrom 290 mg of the peptide resin by stirring for 180 min at roomtemperature with a mixture of 3 ml TFA, 225 mg phenol, 75 μlethanedithiol, 150 μl thioanisole and 150 =82 l H₂ O. The cleavagemixture was filtered, the remaining resin washed with 1 ml TFA and thefiltrate was concentrated to approximately 1 ml by a stream of nitrogen.The crude peptide was precipitated from this oil with 50 ml diethylether and washed 3 times with 50 ml diethyl ether. The precipitate wasthen dissolved in 50 ml H₂ O and lyophilized. The resulting powder wasthen stirred with 0.5 ml hydrazine hydrate in 5 ml ethanol for 6 h at 70C. and then diluted with 50 ml H₂ O and lyophilized.

The lyophilized product was dissolved by addition of 2 ml acetic acid, 2ml ethanol and 100 ml H₂ O and purified using procedures similar tothose described in example 1. The yield was 9.43 mg

RP-HPLC analysis using conditions A1 and B1 gave the retention times of23.22 min and 25.05 min respectively.

EXAMPLE 61

H-Aib-Hisψ(CH₂ NH)D-2Nal-D-Phe-Lys-OH

The peptide was synthesized using a procedure similar to the onedescribed in example 5. with the exception that 1050 mg Sasrin resin(2-Methoxy-4-alkoxybenzyl alcohol resin) (Bachem, Bubendorf, Switzerlandcat. # D-1295) with a substitution capacity of 0.87 mmol/g was used andthe protocol used for coupling the first amino acid residue to the resinwas a 4-dimethylaminopyridine catalysed coupling of the preformedsymmetrical anhydride followed by capping of residual --OH groups on theresin with benzoic anhydride. Upon cleavage of 752 mg peptide resin andpurification of 7/10 of the resulting crude product as described inexample 1, a yield of 36.76 mg was obtained.

The final product was characterised as described in Example 1. Theretention time using elution conditions A1 and B1 was found to be 13.90min and 17.42 min, respectively.

EXAMPLE 62 H-Aib-His-N-Me-D-2Nal-D-Phe-Lys-NH₂

The peptide resin H-N-Me-D-2Nal-D-Phe-Lys(Cl-Z)-MBHA resin wassynthesized using a procedure similar to the one described in Example 56from a MBHA resin with a substitution capacity of 0.72 mmol/g. 458 mg ofthis resin was then used for synthesis ofH-Aib-His(Bom)-N-Me-D-2Nal-D-Phe-Lys(Cl-Z)-MBHA by coupling withBoc-His(Bom)-OH and Boc-Aib-OH. The N-methylated peptide was thencleaved from 469 mg of the resulting resin and 1/2 of the crude peptidewas purified and characterized as described in example 7. The yield was23.5 mg.

RP-HPLC analysis using conditions A1 and B1 gave the retention times of17.62 min and 19.95 min respectively.

EXAMPLE 63 (Furfuryl)-Aib-His-D-2Nal-D-Phe-Lys-NH₂

The peptide resin H-Aib-His(Trt)-D-2Nal-D-Phe-Lys(Boc)-Rink resin wassynthesized using a procedure similar to the one described in Example 1from 4-((2,4'-4'-dimethoxyphenyl)-(Fmoc-amino)methyl)-phenoxy resin(Rink resin) (Novabiochem, Bad Soden, Germany. cat. #: 01-64-0013) witha substitution capacity of 0.43 mmol/g.

300 mg of the resulting peptide resin was then stirred with 5 ml 1%acetic acid in DMF and 410 μl of furan-2-aldehyde. 231 mg NaCNBH₃ (85%)was added after 15 min and after 180 min. Stirring was continued for18h. The resin was isolated by filtration and washed with DMF, DCM/MeOH6:4 and with DCM.

The N-alkylated peptide was cleaved from 319 mg of the resulting resin,purified and characterized as described in example 1. The yield was 44.8mg.

RP-HPLC analysis using conditions A1 and B1 gave the retention times of19.45 min and 22.23 min respectively.

EXAMPLE 64N,N-di-(2R-Hydroxy-propyl)-3-aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂

The peptide resin (3-aminomethylbenzoyl)-D-2Nal-D-Phe-Lys(CI-Z)-MBHAresin was synthesized using a procedure similar to the one described inExample 7 from 4-methyl benzhydrylamine (MBHA) resin (BissendorfBiochemicals, Hannover, Germany. cat. #: RMIS50) having a substitutioncapacity of 0.72 mmol/g. 500 mg of the resulting peptide resin was thenstirred with 12.5 ml 1% acetic acid in DMF and 410 μl of2(R)-(tetrahydropyran-2(R,S)-yloxy)propanal. 213 mg NaCNBH₃ (85%) wasadded after 5 min. Stirring was continued for 18h. The resin wasisolated by filtration and washed with DMF, DCM/MeOH 6:4 and with DCM.

The N-di-alkylated peptide was cleaved from 490 mg of the resultingresin, purified and characterized as described in example 7. The yieldwas 20.72 mg.

RP-PLC analysis using conditions A1 and B1 gave the retention times of23.40 m i 25.33 min respectively.

EXAMPLE 65 - 110

    __________________________________________________________________________                                    RP-                                                                           HPLC                                                                              RP-                                                                       reten-                                                                            HPLC                                                                      tion                                                                              reten-                                                               Prepared                                                                           time                                                                              tion                                                                 using a                                                                            for time                                                                 procedure                                                                          condi-                                                                            for                                                                  similar to                                                                         tion                                                                              condi-                                                               example                                                                            A1  tion B1                                   Ex.                                                                              Peptide                 no.  (Ex. 1)                                                                           (Ex. 1)                                   __________________________________________________________________________    65 H--Ala--N--Me--(2-aminobenzoy1)--D-2Nal-D--                                                           7    24.47                                                                             26.27                                        Phe--Lys--NH.sub.2                                                         66 (4-Aminomethylbenzoyl)-D-2Nal-D--Phe--                                                                1    22.22                                                                             23.5                                         Lys--NH.sub.2                                                              67 H--His--Ala--D-2Nal-D--Phe--Lys--NH.sub.2                                                             1    19.90                                                                             21.45                                     68 (2S)-(H--Ala--His--D-2Nal-D--Phe--NH)-1,6-di-                                                         8    17.05                                                                             19.07                                        aminohexane                                                                69 4-(H--Ala--His--D-2Nal-D--Phe--                                                                       3    18.65                                                                             20.08                                        NH)butylamine                                                              70 3-(H--Ala--His--D-2Nal-D--Phe--NH)-propyl-                                                            3    18.42                                                                             19.80                                        amine                                                                      71 (3-Aminomethylbenzoyl)-D-hPhe-D--Phe--                                                                7    20.33                                                                             21.95                                        Lys--NH.sub.2                                                              72 (3-Aminomethylbenzoyl)ψ(CH.sub.2 NH)D-                                                            5    14.17                                                                             17.23                                        2Nal-D--Phe--Lys--NH.sub.2                                                 73 (3-Aminomethylbenzoyl)-D-2Nal-D-hPhe-                                                                 7    25.30                                                                             26.58                                        Lys--NH.sub.2                                                              74 (3-Amino-3-methylbutanoyl)--His--D-2Nal-                                                              58   18.12                                                                             20.20                                        D--Phe--Lys--NH.sub.2                                                      75 (3-Aminomethylbenzoyl)-D-2Nal-N--Bzl--                                                                7    25.33                                                                             26.70                                        Gly--Lys--NH.sub.2                                                         76 (2S)-((3-Aminomethylbenzoyl)-D-2Nal-D--                                                               4    22.95                                                                             24.32                                        Phe--NH)-6-aminohexanol                                                    77 (3-Aminomethylbenzoyl)-D-2Nal-D-Thial-                                                                7    22.13                                                                             23.23                                        Lys--NH.sub.2                                                              78 (2S)-(H--Aib--Hisψ(CH.sub.2 NH)D-2Nal-D--Phe--                                                    59   12.83                                                                             17.27                                        NH)-6-aminohexanol                                                         79 (3-Aminomethylbenzoyl)-D-2Nal-D-3Pyal-                                                                1    15.10                                                                             16.87                                        Lys--NH.sub.2                                                              80 (3-Aminomethylbenzoyl)-D-2Nal-D--Phe(4-                                                               7    23.40                                                                             24.63                                        F)-Lys--NH.sub.2                                                           81 (3-Aminomethylbenzoyl)-D-2Nal-D--Phe(4-                                                               7    22.50                                                                             23.90                                        OMe)-Lys--NH.sub.2                                                         82 2-(H--Aib--His--D-2Nal-D--Phe--NH)ethane                                                              3    18.30                                                                             20.47                                     83 H--Aib--Phe--D-2Nal-D--Phe--Lys--NH.sub.2                                                             1    29.25                                                                             30.55                                     84 2-(H--Aib--His--D-2Nal-D--Phe--NH--(1-                                                                3    18.70                                                                             20.80                                        methyl-2-pyrrolidinyl)ethane                                               85 2-(H--Aib--His--D-2Nal-D--Phe--NH)-(2-                                                                3    19.20                                                                             20.83                                        pyridyl)ethane                                                             86 (3-Aminomethylbenzoyl)--D-2Nal-N--Me--D--                                                             1    26.78                                                                             27.88                                        Phe--Lys--NH.sub.2                                                         87 H--Aib--His--D-2Nal-D--Phe--Gly--NH.sub.2                                                             1    20.48                                                                             22.23                                     88 H--Aib--His--D-2Nal-D--Phe--Ala--NH.sub.2                                                             1    21.65                                                                             23.38                                     89 H--Aib--His--D-2Nal-D--Phe--Orn--NH.sub.2                                                             1    18.43                                                                             20.05                                     90 (5-Aminomethylthienyl-2-carbonyl)-D-                                                                  1    22.32                                                                             23.65                                        2Nal-D--Phe--Lys--NH.sub.2                                                 91 H--Aib--His--D-2Nal-D--Phe--D--Lys--NH.sub.2                                                          1    18.50                                                                             20.00                                     92 H--Aib--His--D-2Nal-D--Phe--Dab--NH.sub.2                                                             1    17.75                                                                             19.48                                     93 H--Aib--His--D-2Nal-D--Pheψ(CH.sub.2 NH)Lys--                                                     5    18.57                                                                             20.62                                        NH.sub.2                                                                   94 H--Aib--His--D-2Nal-D--Phe--N--Me--Lys--NH.sub.2                                                      62   18.03                                                                             20.60                                     95 (3-Aminomethylthienyl-2-carbonyl)-D-                                                                  1    23.23                                                                             24.78                                        2Nal--D--Phe--Lys--NH.sub.2                                                96 H--Aib--His--D-2Nal-N--Me--D--Phe--Lys--NH.sub.2                                                      1    21.78                                                                             23.53                                     97 H--Aib--His--D-2Nal-D--Phe--Lys--N(Me).sub.2                                                          3    18.70                                                                             21.07                                     98 (3-Aminomethylbenzoyl)-D-1Nal-D--Phe--                                                                1    22.67                                                                             24.23                                        Lys--NH.sub.2                                                              99 H--Aib--His--D-2Nal-D--Trp--Lys--NH.sub.2                                                             1    18.17                                                                             20.40                                     100                                                                              (2-Pyridylmethyl)-Aib--His--D-2Nal-D--Phe--                                                           63   19.07                                                                             21.73                                        Lys--NH.sub.2                                                              101                                                                              H--Aib-(3-aminomethylbenzoyl)-D-2Nal-D--                                                              7    23.95                                                                             25.38                                        Phe--Lys--NH.sub.2                                                         102                                                                              H--Aib-3Pyal-D-2Nal-D--Phe--Lys--NH.sub.2                                                             1    18.53                                                                             20.38                                     103                                                                              (3R)-Piperidinecarbonyl)-D-2Nal-D--Phe--                                                              1    21.52                                                                             22.97                                        Lys--NH.sub.2                                                              104                                                                              (2-(H--Aib--His--D-2Nal-NH)ethyl)benzene                                                              3    25.55                                                                             27.85                                     105                                                                              (2R-Hydroxypropyl)-Aib--His--D-2Nal-D--                                                               64   18.15                                                                             20.28                                        Phe--Lys--NH.sub.2                                                         106                                                                              (3-Aminomethylbenzoyl)-D-2Nal-D--                                                                     5    22.00                                                                             23.95                                        Pheψ(CH.sub.2 NH)Lys--NH.sub.2                                         107                                                                              (3-Aminomethylbenzoyl)-N--Me--D-2Nal-D--                                                              62   23.27                                                                             24.72                                        Phe--Lys--NH.sub.2                                                         108                                                                              (3-Aminomethylbenzoyl)-D-2Nal-D--Phe--N--                                                             67   22.60                                                                             23.98                                        Me--Lys--NH.sub.2                                                          109                                                                              H--D--Thr--His--D-2Nal-D--Phe--Lys--NH.sub.2                                                          7    17.75                                                                             19.83                                     110                                                                              H--Hyp--His--D-2Nal-D--Phe--Lys--NH.sub.2                                                             7    17.58                                                                             19.37                                     __________________________________________________________________________

The structures of representative peptides of the invention are shownbelow. ##STR17##

EXAMPLE 116

An in-vitro assay using rat pituitary cells was established to study theeffect of different GH secretagogues. The mixed pituitary cell culturewas isolated from the anterior pituitary of male rats and cultured for 3days. After washing the cells were stimulated for 15 min and the amountof GH secreted measured in the culture supernatant.

The isolation of rat pituitary cells was a modification of 0. Sartor etal., Endocrinolog 116, 1985, pp. 952-957. Pituitaries were dischargedfrom 250 g male Sprague-Dawley rats after decapitation. Theneurointermediate lobes were removed and the remainder was placed inGrey's medium supplemented with 0.25% glucose, 2× non essential aminoacid and 1% BSA (isolation buffer). The glands were cut into smallpieces and transferred to a flask containing 3 ml isolation buffer+11.5mg trypsin and 1000 μg DNase and incubated for 35 min at 37° C., 95% ° 2and 70 rotations per min. The fragments were washed 3 times bysedimentation in isolation buffer and aspirated into single cells byusing a pasteur pipet. After dispersion the cells were filtered though anylon filter (160 jum) to remove undigested tissue. The cells werewashed 3 times with isolation buffer supplemented with trypsin inhibitor(0.75 mg/ml) and resuspended in culture medium (DMEM supplemented with25 mM HEPES, 4mM Glutamine, 0.75% sodium bicarbonate, 2.5% FCS, 3% horseserum, 10% rat serum, 1 nM T₃ and 40 gg/L dexamethasone) to a density of2×105 cells/ml. The cells were seeded into microtiter plates, 200μl/well and cultured for 3 days at 37° C.and 8% CO₂.

Following the culture period the cells were washed twice withstimulation buffer (HBSS supplemented with 1% BSA, 0.25% D-glucose and25 mM HEPES) and preincubated for 1 hour. The buffer was then removedand new stimulation buffer containing the peptide was added and theplates were incubated for 15 min at 37° C.and 5% CO₂. The medium wascollected and analyzed for rat growth hormone (rGH) content in ascintillation proximity assay (SPA) as follows (SPA, essentially asdescribed in U.S. Pat. No. 4,568,649, Hart and Greenwalt, Mol.Immunol.16, 1979, pp. 265-269, or Udenfriend et al., Proc.Natl.Acad.Sci. USA 82,1985, pp. 8672-8676).

The rGH assay was performed in OptiPlates (96-well plates) suitable fordirect counting in a Packards TopCount (β-scintillation counter).

Assay Protocol:

40 μl buffer

10 μl sample (incubated stimulation buffer)

50 μl I²⁵ 1-rGH

50 μl rabbit anti-rGH

50 μl SPA reagent (anti-rabbit antibody)

The plates are sealed and placed on a plate shaker for 30 minutesfollowed by 10 hours of incubation and settling at 10-15° C.andcounting.

In the SPA, rGH bound to an anti-GH rabbit antibody (primary antibody)is reacted with a second antibody bound to fluomicrospheres (SPA Type IIRIA available from Amersham). Any radiolabelled rGH which is bound tothe primary antibody will be immobilized on the fluomicrospheres whichwill then produce light. Measurement in a β-scintillation counter makesit possible to calculate the amount of radiolabelled rGH. The amount ofradiolabelled rGH bound to the fluomicrospheres decreases with anincreasing content of rGH in the sample.

    ______________________________________                                                                            E.sub.max                                 Ex.                          EC.sub.50                                                                            (% of                                     No.  Compound                (nM)   GHRP-6)                                   ______________________________________                                        3    H--Ala--His--D-2Nal-DPhe--NH--(CH.sub.2).sub.5                                                        20     100                                            NH.sub.2                                                                 10   H--Ala--Phe--D-2Nal-D--Phe--Lys--NH.sub.2                                                             2      90                                        51   H--Ala--His--D-1Nal-D--Phe--Lys--NH.sub.2                                                             10     85                                        76   (2S)-((3-Aminomethylbenzoyl)-D2Nal-D--                                                                26     65                                             Phe--NH)-6-aminohexanol                                                  83   H--Aib--Phe--D-2Nal-D--Phe--Lys--NH.sub.2                                                             8      75                                        88   H--Aib--His--D-2Nal-D--Phe--Ala--NH.sub.2                                                             11     80                                        104  (2-(H--Aib--His--D-2Nal-NH)ethyl)benzene                                                              58     85                                        ______________________________________                                    

We claim:
 1. A compound of general formula I

    A-B-C--D(--E).sub.p                                        I

wherein p is 0 or 1; A is hydrogen or R¹ --(CH₂)_(q) --(X)_(r)--(CH₂)_(s) --CO--, whereinq is 0 or an integer from 1 to 5; r is 0 or1; s is 0 or an integer from 1 to 5; R¹ is hydrogen, imidazolyl,guanidino, piperazino, morpholino, piperidino or N(R²)--, wherein eachof R² and R³ is independently hydrogen or lower alkyl optionallysubstituted by one or more hydroxyl, pyridinyl or furanyl groups; and X,when r is 1 , is --NH--, --CH₂ --, --CH═CH--, ##STR18## ##STR19##wherein each of which is disubstituted; B is (G)_(t) --(H)_(u) wherein tis 0 or 1;u is 0 or 1; G and H are amino acid residues selected from thegroup consisting of natural L-amino acids or their correspondingD-isomers, and non-natural amino acids and wherein, when both t and uare 1, the amide bond between G and H is optionally replaced with##STR20## ##STR21## each of which is optionally substituted withhalogen, lower alkyl, lower alkyloxy, lower alkylamino, amino orhydroxy; D, when p is 1, is a D-amino acid of formula --NH--CH((CH₂)_(k)--R⁵)--CO--or, when p is 0, D is --NH--CH((CH₂)₁ --R⁵)--CH₂ --R⁶ or--NH--CH((CH₂)_(m) --R⁵)--CO--R⁶, whereink is 0, 1 or 2; 1 is 0, 1 or 2;m is 0, 1 or 2; R⁵ is selected from the group consisting of ##STR22##each of which is optionally substituted with halogen, alkyl, alkyloxy,amino or hydroxy; and R⁶ is piperazino, morpholino, piperidino, --OH or--N(R⁷)--R⁸, wherein each of R⁷ and R⁸ is independently hydrogen orlower alkyl; E, when p is 1, is --NH--CH(R¹⁰)--(CH₂)_(v) --R⁹, whereinvis 0 or an integer from 1 to 8; R⁹ is hydrogen, idazolyl, guanidino,piperazino, morpholino, 1-methylpyrrolidinyl, piperidino, ##STR23##wherein n is 0, 1 or 2, and R¹⁹ is hydrogen or lower alkyl, ##STR24##wherein o is an integer from 1 to 3, wherein the morpholino isoptionally substituted with ethyl or propyl and the 1-methylpyrrolodinylgroup is optionally substituted with ethyl, or R⁹ is N(R¹¹)--R¹², eachof R¹¹ and R¹² is independently hydrogen or lower alkyl, or R⁹ isindependently ##STR25## each of which is optionally substituted withhalogen, alkyl, alkyloxy, amino, alkylamino, hydroxy, or the Amadorirearrangement product from an amino group and a hexapyranose or ahexapyranosyl-hexapyranose and R¹⁰, when p is 1, is selected from thegroup consisting of --H, --COOH, --CH₂ --, CO--R¹³ and --CH₂ --OH,wherein R¹³ is piperazino, morpholino, piperidino, --OH or--N(R¹⁴)--R¹⁵, wherein each of R¹⁴ or R¹⁵ is independently hydrogen andlower alkyl; with the proviso that, when R⁹ is phenyl, indolyl, orhydroxyphenyl, then R¹⁰ is different from --COOH, CH₂ --OH and--CO--R¹³, wherein R¹³ is --N(R¹⁴)--R¹⁵, the amide bond between B and Cor, when t and u are both 0, between A and C being optionally replacedwith ##STR26## or, when p is 1, the amide bond between D and E beingoptionally replaced with ##STR27##
 2. The compound according to claim 1,in which G and H are non-natural amino acids selected from the groupconsisting of 1,4-diaminobutyric acid, amino-isobutyric acid,1,3-diaminopropionic acid, 4-aminophenylalanine, 3-pyridylalanine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,1,2,3,4-tetrahydronorharman-3-carboxylic acid, N-methylanthranilic acid,anthranilic acid, N-benzylglycine, 3-amino-3-methylbenzoic acid,3-amino-3-methyl butanoic acid, sarcosine, nipecotic acid andiso-nipecotic acid.
 3. The compound according to claim 1, wherein pis
 1. 4. The compound according to claim 1, wherein A is hydrogen. 5.The compound according to claim 1, wherein A is R¹ --(CH₂)_(q) --(X)_(r)--(CH₂)_(s) --CO--, wherein R¹ is 3-imidazolyl, q is 2, r is 0 and s is0; or wherein R¹ is NH₂, q is 1, r is 1, X is disubstituted benzenesubstituted in the 1 and 3 positions, and s is 0; or wherein R¹ is NH₂,q is 1, r is 1, X is disubstituted thiophene substituted in the 3 and 2positions, and s is
 0. 6. The compound according to claim 1, wherein,when t is 1, G is Ala, Gly, Aib, sarcosine, nipecotic acid, oriso-nipecotic acid.
 7. The compound according to claim 1, wherein, whenu is 1, H is His, Phe, Tic, 3Pyal, Gly, Ala, Phe(4-NH₂), Sar, Pro, Tyr,Arg, Orn, 3-aminomethylbenzoic acid or D-Phe.
 8. The compound accordingto claim 1, wherein R⁴ is 2-naphthyl.
 9. The compound according to claim1, wherein R⁵ is phenyl.
 10. The compound according to claim 1, whereinv is 2-6, and R⁹ is --NH₂, morpholinopropane, morpholinoethyl or(1-methylpyrrolidinyl)ethane.
 11. The compound according to claim 1,wherein R¹⁰ is --COOH, CH₂ --OH, --H or CONH₂.
 12. A compound fortreating medical disorders resulting from a deficiency in growth hormoneselected from the group consisting ofH-Ala-His ψ(CH₂NH)D-2Nal-D-Phe-Lys-NH₂ H-Ala-Ala-D-2Nal-D-Phe-Lys-NH₂H-His-D-2Nal-D-Phe-Lys-NH₂ (3-(4-Imidazolyl)propionyl)-D-2Nal-D-Phe-Lys-NH₂H-D-Lys-D-2Nal-D-Phe-Lys-NH₂ H-5Apent-His-D-2Nal-D-Phe-Lys-NH₂H-D-Ala-D-2Nal-D-Phe-Lys-NH₂ H-5Apent-D-2Nal-D-Phe-Lys-NH₂(n-Propyl)-His-D-2Nal-D-Phe-Lys-NH₂ H-Ala-3Pyal-D-2Nal-D-Phe-Lys-NH₂-Ala-Phe(4-NH₂)-D-2Nal-D-Phe-Lys-NH₂ H-D-Ala-His-D-2Nal-D-Phe-Lys-NH₂(2-(4-Imidazolyl)acetyl)-D-2Nal-D-Phe-Lys-NH₂ (3-(4-Imidazolyl)acryloyl)-D-2Nal-D-Phe-Lys-NH₂ (3-Aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂ (3-Aminophenylacetyl)-D-2Nal-D-Phe-Lys-NH₂(4-Aminophenylacetyl)-D-2Nal-D-Phe-Lys-NH₂ (3-Aminocrotonoyl)-D-2Nal-D-Phe-Lys-NH₂(4-Piperidino-carboxyl)-D-2Nal-D-Phe-Lys-NH₂ H-Ala-H is-D-2Nal-D-Phe-NH₂(H-Ala-His-D-2Nal-D-Phe-NH)hexane 6-(H-A la-His-D-2Nal-D-Phe-NH)hexylamine 5-(H-Ala-His-D-2Nal-D-Phe-NH)pentylanaineH-Ala-His-D-2Nal-D-Pheψ(CH₂ NH)Lys-NH₂ H-Ala-His-D-2Nal-D-Phe-Lys-OH(2S)-(H-A la-H is-D-2Nal-D-Phe-NH)-6-aminohexanol(2-(H-Ala-His-D-2Nal-D-Phe-NH)ethyl)benzene2-(H-Ala-His-D-2Nal-D-Phe-NH)ethylamine 4-((H-Ala-His-D-2Nal-D-Phe-NH)methyl)benzylamineH-Ala-His-D-2Nal-D-Phe-Lys(maltosyl)-NH₂ H-Ala-His-D-Phe-D-Phe-Lys-NH₂H-Ala-His-D-Trp-D-Phe-Lys-NH₂ H-His-D-2Nal-D-Trp-Lys-NH₂ H-Ala-His-D- 1Nal-D-Phe-Lys-NH₂ H-Ala-Phe-D-2Nal-D-Phe-Lys-NH₂H-Ala-His-D-2Nal-D-Phe-Lys(maltosyl)-NH₂(2R)-(H-Ala-His-D-2Nal-D-Phe-Lys-NH)-3phenylpropylamineH-Ala-N-Me-(2-aminobenzoyl)-D-2Nal-D-Phe-Lys-NH₂(3-(Methylaminomethyl)benzoyl)-D-2Nal-D-Phe-Lys-NH₂(4-(Aminomethyl)benzoyl)-D-2Nal-D-Phe-Lys-NH₂H-His-Ala-D-2Nal-D-Phe-Lys-NH₂ 4-(H-Ala-His-D-2Nal-D-Phe-NH)butylamine3-(H-Ala-His-D-2Nal-D-Phe-NH)propylamine(3-(Dimethylaminomethyl)benzoyl)-D-2Nal-D-Phe-Lys-NH₂ (3-Amino-3-methylbutanoyl)-D-2Nal-D-Phe-Lys-NH₂ (3-Aminomethylbenzoyl)-D-hPhe-D-Phe-Lys-NH₂ (3-Aminomethylbenzoyl)ψ(CH₂NH)D-2Nal-D-Phe-Lys-NH₂ (3 -Aminomethylbenzoyl)-D-2Nal-D-hPhe-Lys-NH₂ (3-Amino-3-methylbutanoyl)-His-D-2Nal-D-Phe-Lys-NH₂ (3-Aminomethylbenzoyl)-D-2Nal-N-Bzl-Gly-Lys-NH₂(2S)-((3-aminomethylbenzoyl)ψ(CH₂ NH)-D-2Nal-D-Phe-NH)-6-aminohexanol (²S)-((³ -aminomethylbenzoyl)-D-2Nal-D-Phe-NH)-6-aminohexanol(3-Aminomethylbenzoyl)-D-2Nal-D-Thial-Lys-NH₂ (² S)-(H-Aib-Hisψ(CH₂NH)-D-2Nal-D-Phe-NH)-6-aminohexanol(3-Aminomethylbenzoyl)-D-2Nal-D-3Pyal-Lys-NH₂ (3-Aminomethylbenzoyl)-D-2Nal-D-Phe(4-F)-Lys-NH₂ (3-Aminomethylbenzoyl)-D-2Nal-D-Phe(4-OMe)-Lys-NH₂(2-Aminomethylphenylacetyl)-D-2Nal-D-Phe-Lys-NH₂(2-Aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂2-(H-Aib-His-D-2Nal-D-Phe-NH)-(4-pyridyl)ethaneH-Aib-Phe-D-2Nal-D-Phe-Lys-NH₂ 2-(H-Aib-His-D-2Nal-D-Phe-NH)-( 1-methyl-2-pyrrolidinyl)ethane2-(H-Aib-His-D-2Nal-D-Phe-NH)-(4-pyridyl)ethane H-Aib-Hisψ(CH₂NH)-D-2Nal-D-Phe-Lys-OH (3-Aminomethylbenzoyl)-D-2Nal-N-Me-D-Phe-Lys-NH₂H-Aib-His-D-2Nal-D-Phe-Gly-NH₂ H-Aib-His-D-2Nal-D-Phe-Ala-NH₂H-Aib-His-D-2Nal-D-Phe-Orn-NH₂(5-Aminomethylthienyl-2-carbonyl)-D-2Nal-D-Phe-Lys-NH₂H-Aib-His-D-2Nal-D-Phe-D-Lys-NH₂ H-Aib-His-D-2Nal-D-Phe-Dab-NH₂H-Aib-His-D-2Nal-D-Pheψ(CH₂ NH)-Lys-NH₂H-Aib-His-N-Me-D-2Nal-D-Phe-Lys-NH₂ H-Aib-His-D-2Nal-D-Phe-Lys-NH₂H-Aib-His-D-2Nal-D-Phe-N-Me-Lys-NH₂(3-Aminomethylthienyl-2-carbonyl)-D-2Nal-D-Phe-Lys-NH₂H-Aib-His-D-2Nal-N-Me-D-Phe-Lys-NH₂ H-Aib-His-D-2Nal-D-Phe-Lys-N(Me)₂(3R)-Piperidinecarbonyl-D-2Nal-D-Phe-Lys-NH₂ (3S)-Piperidinecarbonyl-D-2Nal-D-Phe-Lys-NH₂ (3-Aminomethylbenzoyl)-D- 1Nal-D-Phe-Lys-NH₂ H-Aib-His-D-2Nal-D-Trp-Lys-NH₂(Furfuryl)-Aib-His-D-2Nal-D-Phe-Lys-NH₂(2-Pyridylmethyl)-Aib-His-D-2Nal-D-Phe-Lys-NH₂ H-Aib-(3-aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂H-Aib-3Pyal-D-2Nal-D-Phe-Lys-NH₂ (3S)-Piperidinecarbonyl-D-2Nal-D-Phe-Lys-NH₂(3R)-Piperidinecarbonyl-D-2Nal-D-Phe-Lys-NH -(2-(H-Aib-His-D-2Nal-NH)ethyl)benzeneN,N-di(2R-Hydroxypropyl)-(3-aminomethylbenzoyl)-D-2Nal-D-Phe-Lys-NH₂(2R-Hydroxypropyl)-Aib-His-D-2Nal-D-Phe-Lys-NH₂(3-Aminomethylbenzoyl)-D-2Nal-D-Phe y(CH₂ NH)Lys-NH₂(3-Aminomethylbenzoyl)-N-Me-D-2Nal-D-Phe-Lys-NH₂ (3-Aminomethylbenzoyl)-D-2Nal-D-Pbe-N-Me-Lys-NH₂H-D-Thr-His-D-2Nal-D-Phe-Lys-NH₂H-Aib-His-D-2Nal-N-(phenethyl)-Gly-Lys-NH₂(3-Aminomethylbenzoyl)-D-2Nal-N-(phenethyl)-Gly-Lys-NH₂H-Hyp-His-D-2Nal-D-Phe-Lys-NH₂ H-Aib-His-N-Me-D-2Nal-N-(phenethyl)-Gly-Lys-NH₂H-Aib-His-N-Me-D-2Nal-N-Me- D-Phe-Lys-NH₂ H-Aib-His-D-2Nal-D-Pheψ(CH₂N(Me))Lys-NH₂ 3-(H-Aib-His-D-2Nal-N-Me-D-Phe-NH)morpholinopropane2-(H-Aib-His-D-2Nal-N-Me-D-Phe-NH)-( 1 -methyl-2-pyrrolidinyl)ethane(3R)-Piperidinecarbonyl-N-Me-D-2Nal-N-Me-D-Phe-Lys-NH₂ 3-((Aminomethylbenzoyl)-D-2Nal-N-Me-D-Phe-NH)morpholinopropane2-(H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NH)-( 1-methyl-2-pyrrolidinyl)ethane2-(3R)-Piperidinecarbonyl-N-Me-D-2Nal-N-Me-D-Phe-NH)-( I-methyl-2-pyrrolidinyl)ethane2-((3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me-D-Phe-NH)-( 1-methyl-2-pyrrolidinyl)ethane3-(H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NH)morpholinopropane3-((3R)-Piperidinecarbonyl-N-Me-D-2Nal-N-Me-D-Phe-NH)morpholinopropane3-((3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me-D-Phe-NH)morpholinopropane H-A Aib-His-D-2Nal -N-Me-D-Phe-Hyp-NH₂2-((3-Aminomethylbenzoyl)-D-2Nal-N-Me-D-Phe-NH)-(l-methyl-2-pyrrolidinyl)ethane and2-((3R)Piperidinecarbonyl-D-2Nal-N-Me-D-Phe-NH)-(1-methyl-2-pyrrolidinyl)ethane.
 13. A pharmaceutical compositioncomprising, as an active ingredient, the compound of claim 1 or apharmaceutically acceptable salt thereof together with apharmaceutically acceptable carrier or diluent.
 14. A compositionaccording to claim 13 in unit dosage form, comprising from about 10 toabout 200 mg of the compound of the general formula I or apharmaceutically acceptable salt thereof.
 15. A pharmaceuticalcomposition for stimulating the release of growth hormone from thepituitary, the composition comprising, as an active ingredient, thecompound of claim 1 or a pharmaceutically acceptable salt thereoftogether with a pharmaceutically acceptable carrier or diluent.
 16. Amethod of stimulating the release of growth hormone from the pituitary,the method comprising administering to a subject in need thereof aneffective amount of the compound of claim 1 or a pharmaceuticallyacceptable salt thereof.
 17. The method according to claim 16, whereinthe effective amount of the compound of the general formula I orpharmaceutically acceptable salt or ester thereof is in the range offrom about 0.0001 to about 100 mg/kg body weight per day.
 18. The methodaccording to claim 16, wherein the effective amount of the compound ofthe general formula I or pharmaceutically acceptable salt or esterthereof is in the range of from about 0.001 to about 50 mg/kg bodyweight per day.